DESCRIPTION: (Applicant's Abstract) Opioid peptides are a family of endogenous peptides including dynorphins, enkephalins and endorphins. They cause naloxone-sensitive analgesic effects mediated through opiate receptors. In addition, non-opioid effects are produced possibly mediated through the NMDA-receptor. Opioid peptides attenuate withdrawal symptoms arising from drug abuse and act as modulators of the immune system and of developmental processes. The regulation of these peptides takes place through specific peptides. N-arginine dibasic convertase is one such peptidase that selectively cleaves opioid peptides containing paired basic residues. This peptidase contains a unique 76 amino acid acidic domain containing more than 70% glutamates and aspartate which is thought to represent a regulatory domain. RD convertase is sensitive to inhibition by the polyamines spermine and spermidine. Polyamines, in addition to their role in cell proliferation, have been known to cause or potentiate analgesic effects of morphine. Since NRD convertase is sensitive to polyamines it is possible that some of these effects take place through regulating NRD convertase peptidase activity. The aim of the studies outlined in this proposal is to understand the function of the unique acidic domain of NRD convertase and to study the regulation of this processing endopeptidase by polyamines and cellular proteins. The polyamine binding site of NRD convertase will be mapped by the use of polyamine receptor antagonists site-specifically substituted polyamine homologs and radiolabeled photoaffinity reagents. Mutants of NRD convertase containing changes in the acidic domain will be generated and examined. Conformational changes of NRD convertase as a result of polyamine binding will be assessed by spectroscopic techniques. A specific inhibitor for NRD convertase will be designed synthesized and tested. We will search for cellular proteins that specifically interact with NRD convertase employing the yeast two-hybrid system for screening cDNA libraries with the acidic domain of NRD convertase as a bait and also by complex formation with a catalytically inactive NRD convertase mutant. Any proteins identified as specifically binding to NRD convertase will be examined for their effects on NRD convertase activity Similarly the effect of complexation on the activity of the interacting proteins will be examined.